First report of prevalence and assemblage analysis of Giardia duodenalis in pigs from Guangxi Zhuang Autonomous Region, southern China

Giardia duodenalis is a common intestinal protozoan that can cause diarrhea and intestinal disease in animals and in humans. However, the prevalence and assemblages of G. duodenalis in pigs from Guangxi Zhuang Autonomous Region have not been reported. In this study, a total of 724 fecal samples (201 from nursery pigs, 183 from piglets, 175 from breeding pigs, and 165 from fattening pigs) were obtained in four areas of the region (Nanning, Yulin, Hezhou, and Guigang). The gene of the small subunit ribosomal RNA (SSU rRNA) of G. duodenalis was amplified by nested PCR. The results show that the prevalence of G. duodenalis in pigs was 3.59% (26/724), of which 14 samples belonged to assemblage A (53.85%) and 12 samples belonged to assemblage E (46.15%). The infection rates of G. duodenalis in Hezhou, Yulin, Nanning, and Guigang were 0%, 0.7%, 10.8% and 1.1%, respectively (χ2 = 45.616, p < 0.01); whereas 5.1% of breeding pigs, 6.0% of piglets, 2.4% of fattening pigs, and 1.0% of nursery pigs were infected with G. duodenalis (χ2 = 8.874, p < 0.05). The SSU rRNA-positive samples were amplified by PCR based on the β-giardin (bg), glutamate dehydrogenase (gdh), and triphosphate isomerase (tpi) genes. Ten, eight and seven positive samples were detected, respectively. Based on phylogenetic analysis of the three genetic loci sequences, a multilocus genotyping A1 was found. The findings of this study provide basic data for the development of prevention and control of G. duodenalis infections in pigs and humans in the Guangxi Zhuang Autonomous Region.


Introduction
Giardia spp.are common intestinal parasites, affecting both humans and a wide variety of other animals [13].The parasite was first discovered over 300 years ago by Antonie van Leeuwenhoek, and since then, six distinct Giardia species have been described [9].The six described Giardia species (Giardia agilis, Giardia psittaci, Giardia ardeae, Giardia muris, Giardia microti, and Giardia duodenalis) infect a wide range of animals including birds, amphibians, rodents, and mammals [20].Giardia duodenalis is an important zoonotic parasite that can cause giardiasis [32,35].The life cycle of G. duodenalis is simple.It consists of two key stages: rapidly multiplying trophozoites which attach to intestinal epithelial cells, and cysts with high resistance to environmental degradation, that are released in the feces and spread through the fecal-oral route [11,19,23].Infection with G. duodenalis shows a wide range of clinical symptoms, such as acute or chronic diarrhea, nausea, abdominal pain, vomiting, and weight loss [10,39].Furthermore, giardiasis affects growth, development, and cognitive functions in infected children [13,34].
The comprehension of G. duodenalis through molecular biological analysis has greatly contributed to the understanding of its taxonomy, population genetics, and epidemiology.The small subunit ribosomal RNA (SSU rRNA), b-giardin (bg), glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) genes are four commonly used gene loci in the genotyping of G. duodenalis, but a single gene may not correctly identify G. duodenalis or completely describe its genetic characterization [25,43].Multilocus genotyping (MLG) based on more than three genes is considered to be more reliable than single-locus genotyping in assemblage and sub-assemblage typing of isolates.As a result, it has been widely used to investigate the genotypic diversity of G. duodenalis, and is considered to be useful for detecting and identifying mixed infections by different assemblages of the parasite [13,37].To date, G. duodenalis can be divided into eight assemblages (A-H) based on genetic analysis, with each assemblage exhibiting a distinct host range [5,20,27].Among the assemblages, assemblages A and B can be found in a wide array of mammals, including humans and pigs [13,31].Other assemblages (C-H) exhibit host specificity and narrow host ranges: assemblages C and D are specific to dogs and other canids; assemblage E is found in domestic animals, such as pigs, cattle, and horses; assemblage F has been identified in cats; assemblages G and H are found in rodents and marine animals such as seals, respectively [8,13,26].However, assemblages C-F have also been identified in humans [22,29,35,44].These findings suggest that close contact between humans and animals may lead to human infection with G. duodenalis.
Although some studies have reported prevalence and distribution of G. duodenalis in pigs [1,15,17,18,41], there are no reports on the infection of G. duodenalis in pigs in Guangxi Zhuang Autonomous Region, China.Pigs infected with G. duodenalis may cause malabsorption and weight loss, resulting in a decline in pig production [48].Currently, assemblages A-F have been found in pigs, with assemblage E the preponderant genotype [4].Therefore, this study aimed to investigate the prevalence of G. duodenalis infection and identify the genotypes present in pigs from Guangxi Zhuang Autonomous Region, China.The study provides essential data concerning G. duodenalis infections in pigs in southern China, which can contribute to the development of targeted public health and effective strategies for prevention and control of giardiasis in this area.

Samples collection
From March 2021 to May 2022, a total of 724 fecal samples (201 from nursery pigs, 183 from piglets, 175 from breeding pigs, and 165 from fattening pigs) were collected from four cities (Nanning, Yulin, Hezhou, and Guigang) in Guangxi Zhuang Autonomous Region, China (Fig. 1).Fecal samples were collected directly from the rectum by using sterile gloves.Subsequently, fecal samples were placed in sterile plastic bags marked with the date, type and farm.All samples were promptly transported to the laboratory in cool boxes with ice packs and stored at À80 °C until DNA extraction.

Genomic DNA extraction
Approximately 200 mg of each fecal sample was aseptically transferred to individual 1.5 mL centrifuge tubes.Genomic DNA extraction was performed using an E.Z.N.A. Ò Stool DNA Kit (D4015-02, OMEGA Bio-Tek Inc., Norcross, GA, USA), according to the manufacturer's instructions.The extracted DNA was subsequently stored at À20 °C to maintain its integrity for subsequent analyses.

PCR amplification
All samples were amplified by using nested PCR targeting the SSU rRNA gene, as previously described [2].The PCR reaction mixture comprising 25 lL was prepared and amplified according to the procedure described by Jing et al. [15].All SSU rRNA-positive samples were subjected to amplification using nested PCR targeting the bg, gdh, and tpi genes, as previously described [7,16,36].A positive control (DNA from G. duodenalis stored in the laboratory at À80 °C) and a negative control (distilled water) were included in each PCR assay.Each sample was processed three times at the SSU rRNA gene and each SSU rRNA-positive sample was processed three times at the bg, gdh, and tpi genes.The PCR products were identified by 1.5% agarose gel electrophoresis (Gene Biotechnology International Trade Co., Ltd., Shanghai, China).

Sequencing and phylogenetic analysis
PCR products of positive samples were processed for twodirectional sequencing by Sangon Biotech (Xiamen, China).The obtained DNA sequences were aligned with homologous sequences available in the GenBank database of the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/).The MLG method was only used to analyze the Neighborjoining trees [33] were constructed using Mega 11 software, employing the kimura-2 parameter model.The reliability of these trees was assessed using the bootstrap method with 1,000 pseudoreplicates.

Statistical analysis
The Chi-square test (SPSS 25.0 Inc., Chicago, IL, USA) was used to assess differences in prevalence between regions, feeding stages and farming practices.Differences of p < 0.05 were considered statistically significant, and differences of p < 0.01 were considered extremely significant.

Nucleotide sequence accession numbers
All nucleotide sequences have been submitted to the Gen-Bank database in NCBI and allocated accession numbers as follows: OQ943958-OQ943959 for the SSU rRNA gene, OQ934094-OQ934095 for the bg gene, OQ934096-OQ934101 for the gdh gene, and OQ934102-OQ934104 for the tpi gene.
Sequence analysis of the SSU rRNA gene showed two assemblages (A and E) of G. duodenalis in pigs.Of the 26 SSU rRNA-positive samples, 14 isolates (53.85%, 14/26) belong to zoonotic assemblage A, while the other 12 isolates (46.15%, 12/26) were identified as assemblage E (Table 1).Ten, eight and seven sequences were obtained by amplifying 26 SSU rRNA-positive samples at the bg, gdh, and tpi gene loci, respectively (Table 1).At the bg locus, six and four isolates belong to assemblages A and E, respectively; at the gdh locus, six and two isolates belonged to assemblages A and E, respectively; and at the tpi locus, five and two isolates belonged to assemblages A and E, respectively (Table 1).Notably, only one fecal sample of assemblage A was successfully sequenced at all three gene loci and one MLG A1 was formed (Table 2).
To explore the genetic relationships of the G. duodenalis isolates from pigs, four phylogenetic trees were constructed using the SSU rRNA, bg, gdh, and tpi sequences of the parasite.The results show that the representative isolates at the four gene loci were distributed into assemblages A and E, respectively (Fig. 2).

Discussion
Giardia duodenalis is an intestinal parasitic protozoan that has attracted much attention in recent years.Infection with G. duodenalis is widespread in pigs, affecting individuals of all age groups [24].In this study, four areas of the Guangxi Zhuang Autonomous Region were investigated, and it was found that the positive rate of G. duodenalis was 3.59% in 724 samples.There have been no reports of pigs infected with G. duodenalis in Vietnam, a neighboring region of the region.However, a report by Verle et al. showed that the prevalence of G. duodenalis infection in humans from Vietnam was 3.2% [38].Therefore, the results of this study can provide a reference for Vietnam, and further research is needed to determine whether the cause of human infection with G. duodenalis is related to pigs [38].The infection rates of G. duodenalis in pigs vary around the world, such as 1% (6/633) in Canada, 3.4% (3/90) in Brazil, 14% (120/856) in Denmark, 14.8% (110/745) in Korea, 25.4% (53/209) in Nigeria, and 31.1% (90/289) in Western Australia [1,3,6,12,17,28].In China, the infection rate of G. duodenalis in this investigation was similar to the prevalence in Sichuan Province (3.1%, 11/357) [21]; it was higher than that detected in Hubei Province (0.97%, 8/826), Xinjiang (2.6%, 21/801) and Henan Province (1.7%, 15/897) [15,18,40], but lower than that reported in Guangdong Province (18.04%, 94/521), Fujian Province (26.9%, 195/725), Shaanxi Province (8.0%, 45/560), and Zhejiang Province (10.5%, 13/124) [41,[46][47][48].These differences can be attributed to several factors, including sample conditions, feeding patterns, age groups, testing methods and seasons changes [4].We speculate that the strict prevention and control measures implemented after the African swine fever outbreak in China may have resulted in lower infection rates in this study than the rates reported in other regions of China.Moreover, the increased frequency and intensity of disinfection have played a crucial role in reducing the transmission of pathogens.
Giardia duodenalis zoonotic assemblage A (n = 14, 53.85%) and assemblage E (n = 12, 46.15%) were identified among the 26 samples.Assemblage A was the predominant genotype in this study.However, in Australia, Armson et al. identified 56 G. duodenalis positive samples belonging to assemblage E (37 samples, 64.9%) and assemblage A (19 samples, 33.3%) [3].A study in Denmark found that 13 G. duodenalis positive samples belonged to assemblage E (11 samples, 84.6%) and assemblage A (2 samples, 15.4%) [28].The finding that pig-derived assemblage A isolates have 100% homology with human-derived isolates at the SSU rRNA locus means the possibility of zoonotic transmission in this areas.Notably, we also identified assemblage E, which has been reported to infect humans [14].
To further expand knowledge of the genetic diversity of G. duodenalis in pigs, the sequence characters of the bg, gdh, and tpi genes were analyzed for the 26 SSU rRNA positive samples and the MLGs were characterized in pigs based on data from these three loci.Amplification success rates at the bg, gdh, and tpi loci varied from 26.92% to 38.46%.The genetic loci of Giardia have different substitution rates, leading to different resolution for parasite typing at varied loci [13].Nested PCR protocols based on multiple-copy genes (e.g.SSU rRNA) have higher diagnostic sensitivities than those based on single-copy genes (bg, gdh, and tpi) [45].Therefore, the SSU rRNA-positive samples not amplified at the other three loci may be due to the limited sensitivity of PCR in detecting the single-copy genes.The present study confirmed that more positive samples of G. duodenalis can be amplified based on the SSU rRNA gene.The same problem has been reported in similar studies in pigs in Xinjiang [15].The results showed that one fecal sample of assemblage A was successfully sequenced at all three loci, with one MLG, A1.In Ogun state of Nigeria, 12 SSU rRNA-positive samples were simultaneously amplified at three loci, with four MLGs [1].In Shaanxi Province, eight SSU rRNA-positive samples were simultaneously amplified at three loci, with four MLGs [41].In Fujian Province, six SSU rRNA-positive samples were simultaneously amplified at three loci, with one MLG [46].In Spain, 76 SSU rRNA-positive samples from humans were simultaneously amplified at three loci, with 23 MLGs [42].In Southwestern Iran, 82 SSU rRNA-positive samples from humans were simultaneously amplified at three loci, with two MLGs [30].These results show that the polymorphism of G. duodenalis is different in different regions.
In short, pigs infected with G. duodenalis in Guangxi Zhuang Autonomous Region may be a potential source of infectious cysts that affect humans.Workers on pig farms and in slaughterhouses are at risk of infection with G. duodenalis.Understanding the transmission dynamics between animals and humans is crucial for effective disease control and prevention strategies.Therefore, public health problems caused by G. duodenalis should be investigated.
In conclusion, this study provides the first report of giardiasis in pigs from Guangxi Zhuang Autonomous Region, China.Although a low infection rate of G. duodenalis (3.59%, 26/724) was found in this study, the identification of assemblages A and E, and the predominance of assemblage A, suggest that pigs may play an important role in the transmission of G. duodenalis.The multilocus genotyping results of bg, tpi, and gdh loci showed only one MLG A1.The identification and understanding of the distribution of G. duodenalis in animals, such as pigs, are crucial for the development and implementation of effective prevention and control measures.These findings highlight the importance of continued surveillance and targeted interventions to mitigate the risk of G. duodenalis transmission between animals and humans.The data presented in this study serve as a foundation for future research on G. duodenalis.

Figure 1 .
Figure 1.Map of the sample collection area.The green areas are the four areas where samples were collected in Guangxi Zhuang Autonomous Region, China.

Figure 2 .
Figure 2. The phylogenetic relationships of G. duodenalis isolates were obtained by neighbor-joining analysis.Bootstrap values > 50% from 1,000 replicates are shown as nodes.The black circles represent the sequences obtained in this study.A. Phylogenetic relationships based on SSU rRNA nucleotide sequences; B. Phylogenetic relationships based on bg nucleotide sequences; C. Phylogenetic relationships based on gdh nucleotide sequences; D. Phylogenetic relationships based on tpi nucleotide sequences.

Table 1 .
Investigation of G. duodenalis infection in pigs in Guangxi Zhuang Autonomous Region.